The present invention relates to the administration of Keratinocyte Growth Factor-2 (KGF-2) to increase levels of platelets, fibrinogen, albumin, globulin and total serum protein. Further, the present invention relates to administering KGF-2 to protect or treat the bladder and prostate. Moreover, the present invention relates to administering KGF-2 to stimulate growth of nasal, oral, and esophageal mucosa, lacrimal glands, salivary glands and Goblet cells.
Thrombocytopenia is a condition in which there is an abnormally small number of platelets in the circulating blood (Stedman""s Medical Dictionary, 26th edition, Marjory Spraycar, Editor (1995). Thrombocytopenia results from various causes, but ultimately occurs when platelets are destroyed, sequestered in the body, or not produced. The differential diagnosis of thrombocytopenia is extensive and complex, and there is a significant overlap among disorders (Doyle B, and Porter D. L. A.A.C.N. Clin. Issues 8: 469-480 (1997).
Thrombocytopenia may be caused by a variety of mechanisms including, but not limited to, drug induced hypersensitivity, idiopathic thrombocytopenia purpura (ITP), posttransfusion purpura, neonatal thrombocytopenia, bone marrow deficiencies identified with metastatic tumors to the bone, aplastic anemia, myelofibrosis, acute and monocytic leukemia, microangiopathic hemolytic anemia which includes disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome, prosthetic valve hemolytic syndrome, cancer chemotherapy, Zieve""s syndrome, sepsis, HELLP preclamptic syndrome, megaloblastic anemia due to B21 and folic acid deficiency, infections such as peritonitis (without septicemia), congenital rubella syndrome, HIV-1 virus infections, Epstein-Barr infectious mononucleosis, rheumatoid-collagen diseases such as systemic lupus, hypertension of pregnancy associated with preclampsias, thyrotoxicosis and uremia (Clinical guide to laboratory tests. (3rd ed). Philadelphia, W.B. Saunders Company, 1995; Clinical Laboratory Medicine. Clinical application oflaboratory data. (6th ed.) St. Louis, Mosby, 1995).
Fibrinogen is an abundant plasma glycoprotein that is synthesized in the liver. Thrombin sequentially cleaves fibrinopeptides A and B from the xcex1 and xcex2 chains of fibrinogen to produce fibrin monomer, which then polymerizes to form a fibrin clot which is the final major step in the coagulation process. Mutations have been identified which alter the release of fibrinopeptides from the xcex1 and xcex2 chains of fibrinogen, the rate of polymerization of fibrin monomers, and the sites for fibrin cross-linking. This mutations lead to dysfibrinogenemias which are almost always inherited as autosomal dominant traits. Patients with afibrinogenemia, who have no detectable fibrinogen in plasma or platelets, may have infrequent, mild spontaneous bleeding episodes. (Harrison""s Principles of Internal Medicine 11th edition Eugene Braunwald et. al., Editors (1987)).
Hypofibrinogenemia refers to a condition in which there is an abnormally low concentration of fibrinogen in the circulating blood plasma (Stedman""s Medical Dictionary, 26th edition, Marjory Spraycar, Editor (1995)). Hypofibrinogenemia may be caused by a variety of conditions or afflictions including, but not limited to, abnormal hepatic synthesis such as that associated with acute hepatitis or cirrhosis, and disseminated intravascular coagulation (DIC).
Albumin, the major serum protein, is considered to be responsible for maintenance of normal serum colloid osmotic pressure, transport of certain hormones and maintaining an endogenous source of amino acids (Buehler, B. A. Ann. Clin. Lab. Sci. 8: 283-286 (1978)). Hypoalbuminemia is a condition in which there is an abnormally low concentration of albumin in the circulating blood. The serum albumin level is one of several clinical parameters of the status of general health. There is a marked correlation between low albumin levels and the incidence of morbidity and mortality in hospitalized patients. Therefore, it is not surprising to find that hypoalbuminemia is a common finding among hospitalized patients. Hypoalbuminemia is known to be associated with delayed wound healing. The hypoalbuminemic state interferes with the normal functioning of the gastrointestinal tract. Qualitative changes in the albumin molecule which occur in renal disease may damage the nephron. Low serum albumin levels may adversely affect the coagulation system (Doweiko, J. P., and Nompleggi, D. J. J.P.E.N. J. Parenter. Enteral. Nutr. 15: 476-483 (1991)).
Hypoalbuminemia can be caused by a variety of afflictions or conditions including, but not limited to, hemorrhages, bums, exudates, rheumatic diseases, granulomatous processes, most bacterial infections, viral infections accompanied by tissue destruction, tissue necrosis, vasculitis, ulcerative bowel disease, serositis, subacute bacterial endocarditis, parasitic infestations, acute and chronic liver disease, amyloidosis, malnutrition, malignancy, congestive heart failure, constrictive pericarditis, cardiac valvular disease, nephrotic syndrome, trauma and crush injuries, gastrointestinal and lymphatic fistulae, and protein-losing gastroenteropathies (Clinical guide to laboratory tests. (3rd ed). Philadelphia, W.B. Saunders Company, 1995; Clinical Laboratory Medicine. Clinical application of laboratory data. (6th ed.) St. Louis, Mosby, 1995).
Globulin is the name for a family of proteins precipitated from plasma or serum by half-saturation with ammonium sulphate. Globulins may be fractionated by solubility, electrophoresis, ultracentrifugation, and other separation methods into many subgroups, the main subgroups being xcex1-, xcex2-, and xcex3-globulins. These differ with respect to associated lipids or carbohydrates and in their content of many physiologically important factors. Globulins include immunoglobulins in the xcex2, and xcex3 fractions, lipoproteins in the xcex1 and xcex2 fractions, gluco- or mucoproteins (orosomucoid, haptoglobulin), and metal binding and metal transporting proteins (such as transferrin, siderophilin, ceruloplasmin). Other substances found in globulin fractions are: macroglobulin, plasminogen, prothrombin, euglobulin, antihemophilic globulin, fibrinogen, and cryoglobulin (Stedman""s Medical Dictionary, 26th edition, Marjory Spraycar, Editor (1995)).
Certain reasonably predictable changes take place in plasma protein levels in response to acute illness. Hypoglobulinemia refers to an abnormally low concentration of globulin in the circulating plasma. Hypoglobulinemia may result from a variety of conditions or afflictions including, but not limited to, alpha-1 antityrpsin deficiencies, severe liver disease, estrogen therapy, megaloblastic anemia, hypogammaglobulinemia and aggammaglobulinemia (Clinical guide to laboratory tests. (3rd ed). Philadelphia, W.B. Saunders Company, 1995; Clinical Laboratory Medicine. Clinical application of laboratory data. (6th ed.) St. Louis, Mosby, 1995).
A decrease in total serum protein is associated with protein loss (protein-losing gastroenteropathies, acute burns, nephrotic syndrome) and decreased synthesis of protein (chronic liver disease, malabsorption syndrome, malnutrition, and agammaglobulinemia).
Hemorrhagic cystitis is a syndrome associated with certain disease states as well as exposure to drugs, viruses, and toxins. It manifests as diffuse bleeding of the endothelial lining of the bladder. Treatment includes intravesical, systemic, and nonpharmacologic therapies (West, N. J. Pharmacotherapy 17: 696-706 (1997)).
Loss of the ability to produce adequate amounts of saliva and tears is a major clinical problem affecting millions of people and there are few therapeutic options for these sufferers. Patients with xerostomia, or dry mouth, have difficulty swallowing, have painful cracks in their mouths, and experience a decrease in their ability to taste. This condition may be caused by Sjogren""s syndrome, as a secondary event to radiation used with patients with head and neck tumors, and to drugs. Patients with Sjogren""s syndrome sometimes have keratoconjunctivitis sicca or dry eye. This condition may be caused by damage to the lacrimal gland due to Sjogren""s syndrome, sarcoidosis, aging, HIV infection, burns, etc. Patients experience irritation, blurring of vision, burning, pain, and increased risk of infections. Millions of patients suffer with Sjogren""s syndrome and therapy is sub-optimal.
As is the case with xerostomia, patients with keratoconjunctivitis sicca have few therapeutic options. At this time there are approximately 10 million patients in the US which require artificial tear preparations (Lemp, M. A, Adv. Exp. Med. Biol. 438:791-803 (1998)). There are no treatments available at this time to stimulate replacement of the cells in the salivary and lacrimal gland cells.
Sinus infections usually occur in the setting of upper respiratory tract infections, allergies, or anatomic defects (within the sinuses or nasal septum) and may lead to symptoms of headache, facial pain, fever, and purulent rhinorrhea. (Evans K L. Drugs 56(1): 59-71 (1998)). The symptoms of nasal allergy and chronic ethmoid sinusitis overlap and treatment failure in allergic patients may suggest possible chronic sinusitis. Chronic ethmoidal sinusitis may be the leading cause of rhinorrhea and nasal obstruction in patients with perennial allergies. (Bertrand et al., Acta Otorhinolaryngol Belg 51(4):227-237 (1997)). Acute sinusitis tends to occur in patients with a history of rhinitis, which may be allergic or non-allergic in origin. Patients with anatomic variants (Evans K L. Drugs 56(1): 59-71 (1998)) and cystic fibrosis (Brihaye et al., Acta Otorhinolaryngol Belg 51(4):323-337 (1997); Ramsey et al., J. Allergy Clin. Immunol. 90(3Pt2):547-552 (1992); Davidson et al., Laryngoscope 105(4Pt1):354-358 (1995); Jones et al., Int. J. Pediatr. Otorhinolaryngol. 28(1):25-32 (1993)) are also at high risk for developing sinusitis. In particular, it is known that occlusion of the sinus ostia starts the cycle of events, which lead to and sustain sinusitis. (Reilly J. S. Otolaryngology Head and Neck Surgery 103(5):856-862(1990)). The goals of treatment for both acute and chronic sinusitis are to control the rhinitis, improve ventilation to the sinuses, and to improve the function of the sinuses for clearance of secretions. (Evans K L. Drugs 56(1): 59-71 (1998)).
Surgical treatment of the nasal sinuses is only considered when medical measures fail to control the sinusitis. The goals of treatment are identical: to improve ventilation and to facilitate or restore sinus drainage. An additional goal is that surgical treatment may sometimes improve the penetration of topical drugs into the sinus. Up to 250,000 procedures are performed each year in the United States. However, in the course of performing sinus surgery, the mucosal surface is stripped away or damaged. In cases where the surgery has been extensive, the underlying bone within the sinus may be exposed for periods of up to 6 months before the mucosa is fully reconstituted and recovery of ciliary density may require up to 2 years to achieve. Delayed re-epithelialization of the nasal sinuses following surgery is believed to be associated with increased risk of infection, scarring (subepithelial fibrosis) leading to recurrent disease, and cyst formation. After 2 years of follow up approximately 2-5% of patients will have recurrent disease and up to 15% may require surgical revision. (Evans K L. Drugs 56(1): 59-71 (1998)).
Thus, there is clearly a need in the art for therapeutic proteins capable of increasing levels of platelets, fibrinogen, albumin, globulin and total serum protein. Further, there is a need for therapies capable of treating or protecting against damage caused by cystitis. Moreover, there is a need for stimulating the proliferation of cells in the salivary and lacrimal glands; and stimulating re-epithelialization of the sinuses and the growth of nasal mucosa in the nasal sinuses after surgery.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the keratinocyte growth factor (KGF-2) having the amino acid sequence is shown in FIG. 1 [SEQ ID NO:2] or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 75977 on Dec. 16, 1994. The nucleotide sequence determined by sequencing the deposited KGF-2 clone, which is shown in FIG. 1 [SEQ ID NO:1], contains an open reading frame encoding a polypeptide of 208 amino acid residues, including an initiation codon at positions 1-3, with a predicted leader sequence of about 35 or 36 amino acid residues, and a deduced molecular weight of about 23.4 kDa. The amino acid sequence of the mature KGF-2 is shown in FIG. 1, amino acid residues about 36 or 37 to 208 [SEQ ID NO:2].
The polypeptide of the present invention has been identified as a member of the FGF family, more particularly the polypeptide has been identified as KGF-2 as a result of amino acid sequence homology with other members of the FGF family.
In accordance with one aspect of the present invention, there are provided novel mature polypeptides which are KGF-2 as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding human KGF-2, including mRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof, and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with another aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques through the use of recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of KGF-2 proteins, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a human KGF-2 nucleic acid sequence.
In accordance with yet a further aspect of the present invention, there is provided therapeutic methods such as increasing platelet levels by administering KGF-2 for the purpose of alleviating thrombocytopenia. KGF-2 can also be used to increase the levels of plasma fibrinogen which may be found in conditions of abnormal hepatic synthesis (such as that associated with acute hepatitis or cirrhosis) and disseminated intravascular coagulation. KGF-2 can further be used to increase levels of serum albumin in patients with hypoalbuminemia. KGF-2 can also be used to increase the levels of serum globulin found in patients with hypoglobulinemia. KGF-2 can also be used to increase the levels of total serum protein in patients with protein loss and decreased protein synthesis.
KGF-2 can further be used to inhibit toxic effects on the prostate and bladder and to reduce the extent of ulceration caused by cystitis.
KGF-2 can also be used to stimulate the growth of sinus epithelia, nasal mucosa, lacrimal glands and salivary glands.